The present invention relates to p53 binding areas (regions) on a CD95 receptor DNA and to the use of the p53 binding regions for influencing apoptosis and/or for identifying substances suitable for this purpose.
p53 is a tumor suppressor which is induced in the case of DNA damage. It then activates target genes so as to achieve growth stand-still in the cells having DNA damage followed by the repair of the DNA damage or death of the cells. The latter is due to apoptosis.
A chemotherapy is to cause DNA damage in tumor cells. This damage shall then lead to the induction of p53 and ultimately to the death of the tumor cells. However, it shows frequently that certain tumor cells are resistant to chemotherapeutic agents or become resistant thereto after a short treatment duration. The reason why this is the case is not really known thus far.
Therefore, it is the object of the present invention to provide a product by which the resistance to chemotherapeutic agents can be investigated and optionally influenced.
According to the invention this is achieved by the subject matters defined in the claims.
The present invention is based on applicant""s insights that the induction of p53 by chemotherapeutic agents directly activates apoptosis. In particular, applicant found that p53 activates CD95-mediated apoptosis in that p53 induces both the expression of the CD95 ligand and that of the CD95 receptor. Applicant also found that p53 binds to CD95 receptor DNA via p53 binding regions. He also identified such bindings regions in intron 1 and/or the promoter of the CD95 receptor DNA. Moreover, applicant recognized that resistance to chemotherapeutic agents may be due to the fact that p53 can no longer bind to the above p53 binding regions (cf. Table 1 and FIGS. 1-6).
According to the invention applicant""s insight are used to provide a p53 binding region of a CD95 receptor DNA.
The term xe2x80x9cp53 binding regionxe2x80x9d comprises any region of a CD95 receptor DNA to which a p53 may bind and activate the CD95 receptor DNA, i.e. may induce it to transcribe. The term xe2x80x9cp53xe2x80x9d comprises p53 in wild-type form as well as p53 in modified form which still has the above function. A p53 binding region according to the invention may be identified and provided by common methods. It is favorable to cleave a CD95 receptor DNA (cf. Behrmann, I. et al., Eur. J. Immunol. 24 (1994), 3057-3962) by Sau 3A1 and insert the fragments in the BamHI site of pBlueScript II KS+. The cloned CD95 receptor DNA fragments are inserted in DNA binding experiments which use cell extracts from the tumor cells, e.g. H1299, Hep3B, HepG2 or Huh7, which had been transfected beforehand with a p53-coding expression vector, e.g. pCMVp53wt. Bound DNA fragments are fused with a reporter DNA, e.g. luciferase DNA. This may be made e.g. in the expression vectors pGL3-Basic (Promega company) or pTATA-LUC (Wirth, T., Wurzburg, Germany). Resulting expression plasmids are tested in luciferase activity tests for their capacity of being activable.
In a preferred embodiment, a p53 binding region comprises the sequence of FIG. 4, (p53 Be sequence)(SEQ ID NO. 24) and/or FIG. 5 (SEQ ID NOs. 12, 14 and 16)(one or more of the p53 Be sequences) or a sequence differing therefrom by one or more base pairs. The expression xe2x80x9ca sequence differing by one or more base pairsxe2x80x9d comprises a sequence of a CD95 receptor DNA which hybridizes with the DNA of FIG. 4 (SEQ ID NOs. 24 and 32 ) and/or FIG. 5 (SEQ ID NO. 12, 14 and 16 ) and to which a p53 may bind and which may activate the CD95 receptor DNA. The sequence may differ from the DNA of FIG. 4 and/or FIG. 5 by additions, deletions, substitutions and/or inversions of one or more base pairs. The expression xe2x80x9chybridizationxe2x80x9d refers to hybridization under common conditions, in particular at 20xc2x0 C. below the melting point of the sequence.
In a particularly preferred embodiment a p53 binding region comprises the sequence of FIGS. 7, (SEQ ID NO. 2), 8 (SEQ ID NO. 3), 9 (SEQ ID NO. 4), 10 (SEQ ID NO. 1), 11 (SEQ ID NOs. 6, 7, 8, and 9), 12 (SEQ ID NOs. 11, 13, 15, 17 and 19) or 13 (SEQ ID NOs. 25, 27, 29 and 31), the sequences of FIGS. 11, 12 and 13 being variations of the sequences of FIGS. 8, 9, and 10, respectively. Furthermore, the sequences of FIGS. 7, 8, 9 and 10 are explained in FIG. 14.
A p53 binding region according to the invention may be present as such or in combination with any other DNA. For example, a p53 binding region according to the invention may be present in a vector, optionally in combination with a reporter DNA, e.g. luciferase DNA. Preferred combinations are the DNA constructs CD95 (Ps)-LUC, CD95 (P)-LUC, CD95 (I+SV)-LUC, CD95 (Ps+I)-LUC, p1139, p1140, p1141, p1142, p1140 IMI, p1140 IMII, p1140 IMIII, p1140 IMIV, p1141 IMIJI, p1141 1p53 , p1141 2p53 , p1141 3p53 , p1141 xcex94BgI, p1141 xcex94Spe, p1141 xcex94Mph, p1142 TAG, p1142 IMIII, p1142 xcex94Bg1, p1142 xcex94Spe and p1142 xcex94Mph, in which a p53 binding region according to the invention is present in the expression vectors pGL3-Basic and/or pTATA-LUC. As to the DNA constructs CD95 (Ps)-LUC, CD95 (P)-LUC, CD95 (I+SV)-LUC, CD95 (Ps+1)-LUC, reference is made to Example 3 and FIG. 6. The DNA constructs p1139, p1140, p1141, p1142, p1140 IMI, p1140 IMII, p1140 IMIII, p1140 IMIV, p1141 IMIIJ, p1141 1p53 , p1141 2p53 , p1141 3p53, p1141 xcex94Bgl, p1141 xcex94Spe, p1141 xcex94Mph, p1142 TAG, p1142 IMJI, p1142 xcex94Bgl, p1142 xcex94Spe, and p1142 xcex94Mph, in which a p53 region according to the invention is present in the expression vectors pGL-3Basic and/or pTATA-LUC. As to the DNA constructs CD95(Ps)-LUC, CD95(P)-LUC, CD95(I+SV)-LUC, CD95(Ps+1)-LUC, reference is made to Example 3 and FIG. 6. The DNA constructs p1139, p1140, p1141, p1142, p1140 IMI, p1140 IMII, p1140 IMIII, p1140 IMIV, p1141 IMIIJ, p1141 1p53, p1141 2p53, p1141 3p53, p1141 xcex94Bgl, p1141 xcex94Spe, p1141 xcex94Mph, p1142 TAG, p1142 IMJI, p1142 xcex94Bgl, p1142 xcex94Spe, and p1142 xcex94Mph contain the sequences indicated in FIGS. 7 (SEQ ID NO. 2), 8 (SEQ ID NO. 3), 9 (SEQ ID NO. 4), or 10 (SEQ ID NO. 1), i.e. p53 binding regions or variations thereof (cf. FIG. 11 (SEQ ID NOs. 6, 7, 8 and 9), 12 (SEQ ID NOs. 11, 23, 25 17 and 19) and 13 (SEQ ID NOs. 25, 27, 29, and 31)). The DNA constructs p1139, p1140, p1141 and p1142 are preferred and were deposited with DSMZ (Deutsche Sammiung fur Mikroarganismen und Zellen [German-type collection of microorganisms and cells]) on Sep. 24, 1999, i.e. p1139 under DSM 3075, p1140 under DSM 13062, p1141 under DSN 13063 and p1142 under DSM 13064.
A further subject matter of the present invention is a kit comprising a p53 binding region according to the invention (a) and common auxiliary ingredients (b), such as buffers, solvents, carriers, controls, etc. One or more representatives of the p53 binding region may be present. The above explanations also apply correspondingly.
The present invention enables mechanisms resulting when DNA is damaged to be investigated on a molecular level. Such mechanisms comprise the response of the cells to eliminate the DNA damage or to kill themselves. The latter is an apoptotic process. The present invention enables mechanisms resulting in a chemotherapy to be investigated. In particular, it is possible to investigate the cause of resistances to chemotherapeutic agents. For example, it can be determined by means of a p53 binding region according to the invention whether tumor cell-derived p53 is still capable of inducing apoptosis.
The present invention is also suitable to identify and provide substances capable of influencing apoptosis. This influence may be an induction or an inhibition. For this purpose, it is favorable to introduce into cells a p53 binding region according to the invention in combination with a reporter DNA, add thereto the substances to be identified and select them for the transcription-activating or transcription-inhibiting effect of the substances. p53 binding regions may be activated or inhibited in a CD95 receptor DNA by means of these substances and therefore induce or inhibit apoptosis.
Thus, the present invention provides products or means serving for influencing apoptotic processes. This is of great significance, since apoptotic processes are modified in many diseases. For example, the apoptosis rate of viral, liver and neurodegenerative diseases is increased whereas it is lowered in autoimmune and tumoral diseases. Thus, the present invention is the possibility of therapeutically influencing these diseases. An application in a diagnostic respect is also useful, in particular if a p53 gene therapy is carried out in connection with the above-mentioned diseases and the vectors used for this purpose are tested for effectiveness, availability, etc., by means of the vectors according to the invention.